Abstracts 309

ZOcc-OH-cholesterol (20a) and 22R-OH-cholesterol (22R) yielded pregnenolone + isocaproaldehyde. 25-OH-chol- ester01 (25-OH) formed pregnenolone f malonic dialdehyde facetone. AG (~~g~~) fully blocked pregnenolone formation from cholesterol and 25-OH. while side-chain cleavage of 420-*2, 20a% and 22R was only partially inhibited. AG therefore exerts its main action on the re- action cholesterol + d 20-22. It is highly probable, that in CLAH this step is blocked. 25-OH in the presence of AG yields mainly _ 3p-OH-cholenic aldehyde -(CA) + acetone while 20a oartiallv vielded 20a.25-di-OH-cholesterol which was slowl; converied into 20:hydroxylated CA. Isolated rat adrenal cells (stimulated with 1 mU ACTH/ml) were incubated with AG (20 ~g/ml). Addition of 2%OH partially inhibited corticosterone production. Without AG,25-OH has a stimulating effect. We propose the hypothesis, that abnormal compounds like CA are responsible for the severity of CLAH.

55. On the unique status of chdesterol 2Oa-hy~o~roxi~ in steroid metabolism SMITH, L. L. and TENG, ,I. I., Division of Bio- chemistry, University of Texas Medical Branch, Galveston, Texas 77550, U.S.A.

Our prior demonstration of the rearrangement of cholesterol 20cc-hydroperoxide to cholest-S-ene-3/$20c(,22R-triol by bovine adrenal cortex mitochondria suggested the inter- mediacy of the 20a-hydroperoxide in pregnenolone bio- synthesis from cholesterol. Additional studies of C,,-, C 21-t and C,,-hydroperoxide metabolism in mammalian, plant, and microbial systems failed to provide other examples of the hydroperoxide-diol rearrangement, re- duction to the co~espon~ng alcohol being commonly encountered. Formation of the 2Oa-hydro~roxide by rat adrenals and of cholesterol 7a- and 7~-hydroperoxides by rat liver has been observed, but enzymic hydroperoxide formation is not readily distinguished from nonenzymic peroxidation. Ethyl linoleate appears to stimulate 20x- hydroperoxide formation in rat adrenal incubations and markedly stimulates 7r- and 7P-hydroperoxide formation in incubations of soybean lipoxygenase or rat liver micro- somes. The status of cholesterol 20a-hydroperoxide is unique as regards its metabolic rearrangement to a vicinal diol implicated in steroid hormone biosynthesis. (Supported by Robert A. Welch Foundation and U.S. Public Health Service Grant HL-10160).

56. Cholesterol side ehaln cleavage in microsomes and mit~h~d~a from corpora lutea COOK. B., STRUTHERS, A. D., DENHOLM, R. B. and TAYLOR, PATRICIA D., University Department of Steroid Biochemistry, Royal Infirmary, Glasgow, G4 OSF, Scotland

Adrenals effect cholestrol side chain cleavage (SCSS) only in mitochondria. This is not true in corpora lutea (CL) but luteal microsomal fractions have been little investigated. CL from pigs, sheep or cows were homogenized; nuclear, mitochondrial, microsomal and cytosol fractions were pre- pared by ultracentrifugation. Fractions were incubated for up to one hour in the presence of malate or succinate and an NADPH generating system, and cholesterol, preg- nenolone and progesterone were determined by gas-liquid chromatography. CSCC activity was confined to micro- somal and mitochondrial fractions and the specific activities

(Pg progesterone/mg protein) of the CSCC complex did not differ significantly between mitochondria and microsomes for any species. Under our incubation conditions, pro- gesterone was produced rather than pregnenolone, regard- less of cell fraction or species. Some ~to~hond~al preparations were examined in a IO-55% sucrose gradient using an MSE HS zonal rotor. Mitochondria were homo- geneous in size; protein concentration, cytochrome C oxidase activity and CSCC activity were well correlated. We conclude that, in luteal cells, mitchondria and endo- plasmic reticulum are equally important in CSCC and. if LH controls progesterone biosynthesis, both fractions should be responsive to the ultimate effector of the gonadotrophin.

3B 1. Steroid biosynthesis: Adrenal Cortex-I

57. Alte~ative pathways of cortieosteroid synthesis in rat adrenals LOMMER, D., DIEDRICHSEN, G. and SINTERHAUF, K., 1 Department of Medicine, University of Mainz, D-65 Mainz, W-Germany

After incubation of rat adrenal quarters with ‘H-acetate, specific radioactivities of cholesterol, pregnenolone and progesterone were 10-100 times lower than those of 1 I-desoxycorticosterone (DOC) and corticosterone (B). ACTH decreased specific radioactivities of cholesterol bv factors of 3-8, but it did not alter those of B and it increased those of DOC 2-3 fold. It seems to be unlikely, therefore that 3H-acetate had been incorporated into DdC and B% cholesterol, pregnenolone and progesterone. Specific radio- activities of cholesterol analyzed separately in mitochond~a and in the remaining cell fraction were identical. This does not support the hypothesis that only a small pool of highly labelled cholesterol (which should be expected within the mitochondria) serves as steroid precursor. 21-OH-pregneuo- .._- lone, the only alternative to progesterone as direct pre- cursor of DOC. was 30-50 (control) and 3-9 (ACTH) timnc _.I.,”

higher in specific radioactivity than DOC and B. Under the influence of “triparanol” (l-(p-diethylaminoethoxyphenyl)- I-(p-tolyl)-2-(p-chlorophenyl)-ethanol) which is known to inhibit the step “desmosterol-cholesterol”, specific radio- activities of cholesterol decreased to f, of the control values. In contrast, there were only slight alterations in the specific radioactivities of 21-OH-pregnenolone, DOC and B. These data strongly suggest that in rat adrenals DOC and B can be synthesized from acetate via alternative pathways not including cholesterol, pregnenolone and progesterone as intermediates, in which 21-OH-pregnenolone may be the direct precursor of DOC.

58. Reciprocal interactions of progesterone and 17a- hydroxyprogesterone as exogenous substrates of rat adrenal 21-hydroxylase ORTA, Z. DE M. and DOMINGUEZ, 0. V., Division of Biochemistry, Department of Scientific Research (IMSS), Mexico City, D.F., Mexico

Due to the small concentration and activity of 17a- hydroxylase present in the rat adrenal, the main corticoids secreated in the rat are DOC, B,, A,, 18-OH-DOC and aldosterone, formed directly from progesterone 1). Because of the limited amounts of 17~-OH-progesterone (II) avail- able, the biosynthesis of S,. Fk and E, is restricted. Since